The present invention relates to the field of biology, e.g., describing compositions which interact in cell signaling. The invention provides various compositions and methods directed to protein interactions occurring in the signal transduction pathway, e.g., compositions which include variants of human Jak3, a protein exhibiting tyrosine kinase-like structure, and which functions in regulating intracellular signaling. In particular, it provides agonists and/or antagonists of said proteins.
Cytokines are molecules that mediate differentiation or other signals typically between the circulating cell components of the mammalian circulatory system. Cytokines function through receptors, many of which have been characterized. See, e.g., Aggarwal and Gutterman (eds. 1991) Human Cytokines: Handbook for Basic and Clinical Research, Blackwell, Oxford.
Binding of cytokines to their receptors induces a cascade of intracellular signaling events that results in increased tyrosine phosphorylation of specific target proteins, and initiates a program of altered gene expression and proliferation. See e.g., Leonard and O""Shea (1998) Annu. Rev. Immunol. 16:293-322. Cytokine receptors typically lack intrinsic tyrosine kinase activity and signal via non-receptor tyrosine kinases of the Jak family that associate with the cytoplasmic domains of receptor chains. The paradigm for ligand-induced signaling is that trans-phosphorylation and activation of receptor-associated Jak kinases occurs upon ligand dependent receptor subunit aggregation. These activated kinases phosphorylate tyrosine residues of the receptor chain subunits, providing docking sites for src-homology (SH2) domains of signal transducers and activators of transcription (STATs), as well as other signaling molecules, e.g., the adapter molecule Shc. STATs are recruited to the phosphorylated receptor, where they are phosphorylated. This allows them to dimerize, translocate to the nucleus, and stimulate the expression of cytokine-inducible genes. While the general model for cytokine-induced signaling has been elucidated, the molecular details of the association of Jak family members with the cytoplasmic domains of receptor chains are not understood.
The functional receptors for the cytokines IL-2, IL-4, IL-7, IL-9, and IL-15 all utilize the common gamma chain (xcex3c) signaling subunit. The importance of signaling through xcex3c is underscored by the fact that specific mutations in either the xcex3c or the Janus kinase that interacts with it, Jak3, result in phenotypically similar Severe Combined Immunodeficiency (SCID). Infants with SCID suffer from severe infections due to reduced levels of T and natural killer (NK) cells, as well as hypogammaglobulinemia. Current preferred treatment typically requires heterologous bone marrow transplantation to replace lost T and NK cell functions.
The Jak family of kinases consists of four known mammalian homologs, each consisting of 1100-1200 amino acids organized into seven Janus homology (JH) domains, based on sequence similarity among the family members. They do not possess classic SH2 or SH3 domains, and, except for the catalytic domains, exhibit little homology to other protein tyrosine kinases (PTKs).
As the IL-2 signaling complex is essential for T cell proliferation and is critically dependent on Jak3 activity, understanding this interaction would be a benefit in effecting processes involving, e.g., graft rejection, rheumatoid arthritis, and autoimmune or inflammatory diseases. The availability of agonists and antagonists to cytokine receptor signaling such as, e.g., IL-2, IL-4, IL-7, IL-9, IL-13, and IL-15 will be used to modulate these processes. The present invention provides these, as well as other proteins, useful, e.g., in determining the structure and mechanisms of immune regulation in a cell via the tyrosine kinases of the JAK family.
The present invention provides compositions that serve as an agonist or antagonist for Jak3 proteins. These agonists and antagonists will be useful in modulating T cell proliferation, and may be important in other hematopoietic or immunological function. In certain circumstances, these molecules will also have in vitro or in vivo therapeutic effects.
The present invention is based, in part, upon a structural analysis of a Jak3 mutation from a patient with autosomal severe combined immunodeficiency disease (SCID).
The mutation is a single amino acid substitution, Y100C, located in Janus homology domain 7 (JH7) that prevents kinase-receptor interaction and also results in a loss of IL-2-induced signaling in B-cells.
In particular, this insight leads to recognition of which specific amino acid residues of Jak3 kinase bind to the IL-2 receptor beta (IL-2Rxcex2) and common gamma (xcex3c) chains, respectively, and initiate biochemical signals critical in controlling immune responses. Additionally, this information, leads to recognition of a region around the Jak3 Y100C mutation which has the ability to interact with xcex3c specifically the proline rich box1 region of the IL-2 receptor. Furthermore, a Jak3/Jak1 chimeric composition containing this region mediates IL-2 responses. This invention embraces natural ligands, e.g., specific mutations (muteins) of the natural sequences, fusion proteins, and chemical mimetics. It is also directed to DNAs encoding such variant proteins. Various uses of these different protein or nucleic acid compositions are also provided.
The present invention provides a polypeptide that: comprises at least three non-overlapping fragments of at least 17 contiguous amino acids selected from residues 70-193 of SEQ ID NO: 2 (human Jak3); interferes with the interaction of a human Jak3 with a human xcex3 common receptor chain; and lacks any 8 contiguous amino acid residue fragment of SEQ ID NO: 1 residues 1-69 and 193-1124. Incertain embodiments: the interaction of the Jak3 with the human xcex3c is determined in a binding assay; the total number of residues of the three fragments is between 55 and 123; the polypeptide is selected from: residues 1-193, 70-193, 1-130, 70-130, or 70-256 of human Jak3; or a residue corresponding to position 98, 99, 100, or 102 of the Jake polypeptide is substituted, either singly or in combination.
In other embodiments, the invention provides a isolated polynucleotide encoding the described polypeptide, or its complement, including variants resulting from the degeneracy of the genetic code or point mutations. Preferably, the variants encode the same activity of the polypeptide; or the polynucleotide is operably linked to a replication or transcription sequence. Also provided is a cell transfected with the polynucleotide. Methods are provided, e.g., comprising screening a library comprising such polypeptides for a polypeptide that interferes with the interaction of a Jak3 with a xcex3c, and identifying those polypeptides which do interfere. In certain embodiments, the screening is inside a cell.
Alternatively, the invention provides a polypeptide that: comprises a sequence matching at least 45 out of 53 of residues 263-315 of SEQ ID NO: 2 (human xcex3c); comprises an amino acid residue substitution corresponding to position 266 and/or 269; and interferes with the interaction of human Jak3 with a human xcex3c. Typically: the matching is at least 48 out of 53; the polypeptide comprises sequence corresponding to residues 263-269; or the residue at position 266 or 269 is substituted with a conservative substitution.
Nucleic acid embodiments include, e.g., an isolated polynucleotide encoding the described polypeptide, or its complement, including variants resulting from the degeneracy of the genetic code or point mutations. Typically, the variants encode the same activity of the polypeptide; or the polynucleotide is operably linked to a replication or transcription sequence. Cells transfected with the described polynucleotide are provided.
The invention provides methods, e.g., comprising screening a library comprising described polypeptides for a polypeptide that interferes with the interaction of a Jak3 with a xcex3c, and identifying those polypeptides which do interfere. Often, the screening is inside a cell.
Yet another embodiment is a method of screening comprising screening a library of compositions for a candidate composition that blocks the interaction of a JH7/JH6 fragment of Jak3 with a box1/box2 fragment of xcex3c. In certain embodiments, the JH7/JH6 fragment of Jak3 interacts specifically with residues 263-315 of human xcex3c; or the box1/box2 fragment of xcex3c interacts specifically with residues 1-193 of human Jak3. Preferably, at least one of the fragments is: a recombinant or synthetic polypeptide; attached to a solid substrate; or detectably labeled. Other embodiments include those where the JH7/JH6 fragment of Jak3 is from human; or the box1/box2 fragment of xcex3c is from human. Preferred embodiments include where the Jak3 from human has a sequence of SEQ ID NO: 1; or the xcex3c from human has a sequence of SEQ ID NO: 2.
I. General
II. Agonists; antagonists
III. Physical Variants
A. fragments
B. post-translational variants
1. glycosylation
2. others
C. species variants
IV. Nucleic Acids
A. mutated natural isolates; methods
B. synthetic genes
C. methods to isolate
V. Antibodies
A. polyclonal
B. monoclonal
C. fragments, binding compositions
VI. Making Agonists and Antagonists
A. recombinant methods
B. synthetic methods
C. natural purification
VII. Uses
A. diagnostic
B. therapeutic
VIII. Kits
A. nucleic acid reagents
B. protein reagents
C. antibody reagents
I. General
The present invention is based, in part, on a structural and mutational analysis of the interaction of Jak3 with cytokine receptor subunit binding sites. In one aspect, the present invention defines minimal regions sufficient to permit interaction between Jak3 and the common gamma chain of the cytokine receptor (xcex3c).
The cytokines IL-2, IL-4, IL-7, IL-9, and IL-15 all bind to a common gamma chain (xcex3c) signaling subunit shared among these various cytokine receptors. The importance of signaling through xcex3c is underscored by the fact that specific mutations in either xcex3c or the Jak3 kinase that interacts with it can produce a resulting disease states phenotypically reminiscent to Severe Combined Immunodeficiency (SCID).
In one aspect the present invention identifies a region of Jak3, including portions of JH6 and JH7, that is sufficient for kinase-receptor contact. It is also demonstrated herein that a Jak3 segment which overlaps two Janus homology domains, JH7 and JH6, defines a region sufficient for Jak3 interaction with the box1 region of xcex3c and for the functional interaction of Jak3 with an IL-2 receptor complex.
The present invention applies mutational analysis to determine minimal regions within Jak3 and xcex3c that permit such interactions. For example, a point mutation (Y100C) within the Janus homology domain 7 (JH7) of Jak3 prevents kinase-receptor interaction resulting in a loss of IL-2-induced signaling in a B-cell line derived from a patient having this defect.
In still another aspect, the invention demonstrates that a Jak3/Jak1 chimera containing only minimal JH6 and JH7 domains of Jak3 interacts with xcex3c and can reconstitute IL-2-dependent responses, including receptor phosphorylation and activation of signal transducer and activator of transcription (STAT) 5b.
The present invention provides various muteins within the Jak3 and xcex3c regions that prevent interaction, e.g., one mutein comprises Janus homology domain 7 (JH7) with wild-type flanking sequence. For example, a particular substitution mutations L98A and I102A prevents Jak3 kinase-receptor interaction.
Insight into the function of this region of Jak3 leads to the recognition of which specific Jak3 amino acid residues bind respectively to the IL-2 receptor beta (IL-2Rxcex2) and common gamma (xcex3c) chains, thereby initiating biochemical signals critical in controlling immune responses. Additionally, knowledge of Jak3 leads to recognition of a region within Jak3 that has the ability to specifically interact with xcex3c.
Furthermore, the invention encompasses a Jak3/Jak1 chimeric composition containing which is sufficient to mediate cell inducing responses to various cytokines, e.g., IL-2, IL-4, IL-7, IL-9, IL-13, and IL-15.
Although the N-terminal half of the Jak kinases, containing the JH7-JH3 domains, has been shown to be required for receptor interaction in several systems, the precise nature of the interaction has not been resolved.
Experiments with Jak2/Jak1 chimeras have suggested that the JH7-JH6 domains of Jak2 are sufficient for physical association to IFN-xcex3R2 by co-immunoprecipitation, and IFN-xcex3-inducible STAT 1 activation in a Jak2-deficient cell line. See, e.g., Kohlhuber, et al. (1997) Mol. Cell. Biol. 17:695-706. Reciprocal experiments using Jak1/Jak2 chimeras suggest that the entire amino-terminal half (JH7-JH3) of Jak1 is required for binding to the IFN-xcex3RI and STAT1 activation. See, e.g., Kohlhuber, et al. (1997) Mol. Cell. Biol. 17:695-706. Deletion analysis has shown that the N-terminal 239 amino acids (JH7-JH6) of Jak2 are indispensable for granulocyte-macrophage colony stimulating factor (GM-CSF) and growth hormone (GH) receptor association. See, e.g., Frank, et al. (1995) J. Biol. Chem. 270,14776-14785; and Zhao, et al. (1995) J. Biol. Chem. 270:13814-13818. In addition, work by Pellegrini and colleagues has demonstrated that the N-terminal half of Tyk2 is required for IFN-xcex1 receptor stabilization in a Tyk2-deficient cell line, and for efficient IFN-xcex1 induced phosphorylation. See, e.g., Gauzzi, et al. (1997) Proc. Nat""l Acad. Sci. USA 94:11839-11844. The relevance of Jak1 and Jak2 structure and function to Jak3 signaling remains uncertain and unpredictable.
In the IL-2 receptor system, a fragment of Jak3 containing JH6 and JH7 could bind xcex3c, but reconstitution of IL-2-induced responses was shown only with a larger region consisting of the JH7-JH4 domains. Thus, although the regions of certain Janus kinases necessary for receptor association are clearly in the amino-terminus, as yet, the shortest regions or discrete domains required, especially in Jak3, has not been defined.
The membrane-proximal region of the cytoplasmic tail of particular cytokine receptors, known as box1/box2 (Murakami, et al. (1991) Proc. Nat""l Acad. Sci. USA 88:11349-11353) have been shown to be required for signaling and are thought to interact with Jak kinases. Specifically, mutational analysis of the proline-rich box1 region of gp130 has revealed a major role for this short 8 amino acid motif in the binding of Jak2. See, e.g., Tanner, et al. (1995) J. Biol. Chem. 270:6523-6530. Although this box1 region of receptors is loosely conserved, each receptor has a proline-rich motif within box1 and mutations within this region can disrupt certain Jak associations. Interestingly, a point mutation (L271Q) in the box1 region of xcex3c disrupts Jak3-xcex3c interaction and causes X-linked combined immunodeficiency. See, e.g., Russell, et al. (1994) Science 266:1042-1045; and Schmalstieg, et al. (1995) J. Clin. Invest. 95:1169-1173. Therefore, although Jaks interact with box1/box2 in the cytoplasmic domains of hematopoietic receptors, the region of Jaks that interact with box1 has not been fully characterized, much less what region of Jak3 interacts with box1 of xcex3c.
The present invention teaches that a single point mutation in Jak3 (Y100C) can lead to a severe phenotype, similar to that found in an autosomal SCID condition, by blocking Jak3 interaction with its cognate receptor xcex3c and hence downstream signaling responses to IL-2. Other point mutations in the JH7 region also block binding of Jak3 with xcex3c. This discovery has been used to further define a region within the JH6-JH7 domain of Jak3 that interacts with box1 of the xcex3c receptor chain. The invention herein also teaches that a Jak3/Jak1 chimera containing the entire JH6-JH7 domain can functionally interact with xcex3c, and can reestablish typical IL-2 signaling. The invention further teaches that the specificity of Jak3 kinase receptor interaction is contained within a defined region of the N-terminus of Jak3 and that the JH6-JH7 domains of Jak3 are necessary for xcex3c mediated signaling responses.
The present invention provides sequence variants, also referred to as mutant proteins (muteins), of Jak3 and xcex3c polypeptides and fragments, e.g., muteins, which serve as agonists and/or antagonists of cytokine signaling. The natural cytokine receptor ligands are capable of mediating various biochemical responses which should lead to biological or physiological responses in target cells, e.g., as described above. In particular, the T cell specificity of these cytokines may lead to important regulation of immune responses.
Physically and structurally, many relevant Janus kinase sequences have been previously described, e.g., Leonard and O""Shea (1998) Annu. Rev. Immunol. 16:293-322; and Duhe, et al. (1998) J Interferon Cytokine Res. 18:1-15. Human Janus kinase 3 (Jak3) sequence is publicly available, e.g., from GenBank (see e.g., Accession Numbers U70065, AC005952, AC005952, AC005759, AC005759, AC005759 and others). Likewise, the sequence of the cytokine receptor gamma common chain xcex3c is publicly available, e.g., from GenBank (see, e.g., Accession Numbers 400048, 2494714, 729281, and 461904).
A mutein candidate antagonist is tested, preferably with a sequence substitution as described, e.g., by measuring Jak3 interaction with its cognate receptor xcex3 common chain. Alternatively, fragments of the two interacting partners may be used. In one assay, a dose response curve of the Jak3 or xcex3c is titrated in the absence or presence of the candidate mutein at a fixed concentration. Typically, the candidate mutein concentration is fixed, preferably within the range of equimolar to the half-maximum of the target Jak kinase, or at a 10-, 100-, or 1000-fold excess of candidate mutein over that half-maximum amount. Typically, the dose response curve of the Jak kinase will shift. The shift will normally be at least one log unit, often two to four log units.
To test partial agonist activity of the candidate mutein, a dose-response curve of the mutein is performed. Assays for biological activity in vitro or in animals are known. Typically, the maximal stimulatory activity of the mutein will be near that of the natural Jak kinase, but partial agonists will show a suboptimal stimulation at saturation, e.g., the maximal activity will plateau at a lesser amount. This amount will often be less than about 90%, preferably less than about 75%, more preferably less than about 50%, and in most preferred embodiments, even less than about 25%. Agonists with an even lesser maximum will still be useful, and often provide the most promising candidates for establishing chemical antagonist properties.
Super-activating agonists will have greater activities.
These activity amounts will often be greater than 110%, typically greater than 120%, preferably greater than 130%, and in most preferred embodiments, greater than 150%, including 2-, 3-, and 5-fold.
Muteins are made typically by site specific mutagenesis at defined positions, but synthetic methods may be applied. As described above, sequences of human Jak3 and xcex3c proteins are available from GenBank (see Accession Number U70065, and others) and the references cited therein. Initially, single and low multiplicity mutagenesis will be constructed, with more complex combinations possible. Significant changes in the nature of solvent exposed residues, e.g., charge reversal or significant size or hydrophobicity change, would be more likely to significantly affect physiological result. Also, significant disruption of the secondary structure, e.g., helical structure, would be also expected to abolish receptor interaction. Conservative substitutions generally would be expected to exhibit similar biological activity.
II. Agonists; antagonists
The process of inhibition or prevention of signaling responses is termed antagonism, and chemical entities with such properties are antagonists. See, e.g., Kenakin (1987) Pharmacological Analysis of Drug-Receptor Interaction Raven Press, NY.
Various classes of antagonists include chemical or neutralization antagonists, competitive antagonists, and noncompetitive antagonists. The chemical or neutralization antagonists typically interact with the interacting partners and prevent continued activation of the signaling pathway and subsequent response, e.g., antibody antagonists which bind to one of the partners and block signaling thereby. Variant proteins are purified and subjected to physical analysis, e.g., CD analysis, to determine whether the protein has a native-like conformation. Its binding behavior is tested, e.g., on cells expressing natural or recombinant Jak3 and xcex3c. The effects of the Jak3 and xcex3c mutein variants may also be tested in knockout mice, e.g., Jak3xe2x88x92/Jak3xe2x88x92 or xcex3cxe2x88x92/xcex3cxe2x88x92 animals.
The competitive antagonists typically are molecules which bind to the same recognition site on Jak3 or xcex3c and block physiological binding. Noncompetitive antagonists bind to a site on Jak or xcex3c distinct from the agonist binding site, and block other signal transduction.
Measurement of antagonist activity and analysis of these results can be performed, e.g., by Schild analysis. See Arunlakshana and Schild (1959) Br. J. of Pharmacol. 14:48-58; Chapter 9 of Kenakin (1987) Pharmacological Analysis of Drug-Receptor Interaction Raven Press, NY.; and Black (1989) Science 245:486-493. Schild analysis with a defined antagonist provides a number of means to evaluate quantity and quality of both interacting partner preparations. For example, analysis of a preparation of partners allows better quality control indications than ELISA or mere bioassay quantitation methods. It provides means to distinguish between a denatured partner, which is more likely to test positive in ELISA assays, and a biologically active signaling molecule.
The described muteins are typically proteinaceous, though a full length natural polypeptide is not necessary. Fragments can be useful, e.g., where they include positions which have been mutated as provided herein.
The term xe2x80x9cpolypeptidexe2x80x9d as used herein includes a significant fragment or segment, and encompasses a stretch of amino acid residues of at least about 8 amino acids, generally at least about 12 amino acids, typically at least about 16 amino acids, preferably at least about 20 amino acids, and, in particularly preferred embodiments, at least about 30 or more amino acids. Virtually full length molecules with few substitutions will be preferred in most circumstances.
Substantially pure typically means that the mutein is free from other contaminating proteins, nucleic acids, and other biologicals derived from the original source organism. Purity may be assayed by standard methods, typically by weight, and will ordinarily be at least about 40% pure, generally at least about 50% pure, often at least about 60% pure, typically at least about 80% pure, preferably at least about 90% pure, and in most preferred embodiments, at least about 95% pure.
The size and structure of the polypeptide should generally be in a substantially stable state, and usually not in a denatured state. The polypeptide may be associated with other polypeptides in a quaternary structure, e.g., to confer solubility, or associated with lipids or detergents in a manner which approximates natural lipid bilayer interactions.
The solvent and electrolytes will usually be a biologically compatible buffer, of a type used for preservation of biological activities, and will usually approximate a physiological aqueous solvent. Usually the solvent will have a neutral pH, typically between about 5 and 10, and preferably about 7.5. On some occasions, one or more detergents will be added, typically a mild non-denaturing one, e.g., CHS or CHAPS, or a low enough concentration as to avoid significant disruption of structural or physiological properties of the ligand.
III. Physical Variants
This invention also encompasses proteins or peptides having sequence variations at positions corresponding to the specified residues, but with substantial amino acid sequence identity at other segments. The variants include species variants and particularly molecules with the same primary sequence but variations beyond primary amino acid sequence, e.g., glycosylation or other modifications.
Amino acid sequence homology, or sequence identity, is determined by optimizing residue matches, if necessary, by introducing gaps as required. See also Needleham, et al. (1970) J. Mol. Biol. 48:443-453; Sankoff, et al. (1983) Chapter One in Time Warps, String Edits, and Macromolecules: The Theory and Practice of Seauence Comparison Addison-Wesley, Reading, Mass.; and software packages from IntelliGenetics, Mountain View, Calif.; and the University of Wisconsin Genetics Computer Group, Madison, Wis.;. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Members of a group do exhibit less dramatic structural differences, which may also be important. Substitutions at designated positions , e.g., at solvent exposed residues, can often be made with homologous residues to retain similar activities, e.g., agonist or antagonist functions. Identity measures will be at least about 85%, usually at least about 95%, preferably at least about 97%, and more preferably at least 98% or more, especially about the particular residue positions identified as appropriate for sequence changes. Regions of particular importance are within about 5 amino acids surrounding the defined positions, more particularly within about 8 amino acids, and preferably within about 11 amino acids adjacent the positions where changes are indicated.
The isolated kinase DNA can be readily modified by nucleotide substitutions, nucleotide deletions, nucleotide insertions, and inversions of nucleotide stretches. These modifications result in novel DNA sequences which encode these proteins having many similar physiological, immunogenic, antigenic, or other functional activity. Enhanced expression may involve gene amplification, increased transcription, increased translation, and other mechanisms.
Janus kinase mutagenesis can also be conducted by making amino acid insertions or deletions. Substitutions, deletions, insertions, or any combinations may be generated to arrive at a final construct. Insertions include amino- or carboxy-terminal fusions. Random mutagenesis can be conducted at a target codon and the expressed mutants can then be screened for the desired activity. Methods for making substitution mutations at predetermined sites in DNA having a known sequence are well known in the art, e.g., by M13 primer mutagenesis or polymerase chain reaction (PCR) techniques. See, e.g., Sambrook, et al. (1989); Ausubel, et al. (1987 and Supplements); and Kunkel, et al. (1987) Meth. Enzymol. 154:367-382.
The present invention also provides recombinant proteins, e.g., heterologous fusion proteins using segments from these proteins. A heterologous fusion protein is a fusion of proteins or segments which are naturally not normally fused in the same manner. A similar concept applies to heterologous nucleic acid sequences.
In addition, new constructs may be made from combining similar functional domains from other proteins. For example, ligand-binding or other segments may be xe2x80x9cswappedxe2x80x9d between different new fusion polypeptides or fragments. See, e.g., Cunningham, et al. (1989) Science 243:1330-1336; and O""Dowd, et al. (1988) J. Biol. Chem. 263:15985-15992.
The phosphoramidite method described by Beaucage and Carruthers (1981) Tetra. Letts. 22:1859-1862, will produce suitable synthetic DNA fragments. A double stranded fragment will often be obtained either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence, e.g., PCR techniques.
xe2x80x9cDerivativesxe2x80x9d of these kinases include amino acid sequence mutants at other positions remote from those specified, glycosylation variants, and covalent or aggregate conjugates with other chemical moieties. Covalent derivatives can be prepared by linkage of functionalities to groups which are found in amino acid side chains or at the N- or C-termini, by standard means. See, e.g., Lundblad and Noyes (1988) Chemical Reagents for Protein Modification, vols. 1-2, CRC Press, Inc., Boca Raton, Fla.; Hugli (ed. 1989) Techniques in Protein Chemistry, Academic Press, San Diego, Calif.; and Wong (1991) Chemistry of Protein Conjugation and Cross Linking, CRC Press, Boca Raton, Fla.
In particular, glycosylation alterations are included, e.g., made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing, or in further processing steps. See, e.g., Elbein (1987) Ann. Rev. Biochem. 56:497-534. Also embraced are versions of the peptides with the same primary amino acid sequence which have other minor modifications, including phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.
Fusion polypeptides between these kinase muteins and other homologous or heterologous proteins are also provided. Many growth factors and kinases are homodimeric entities, and a repeat construct may have various advantages, including lessened susceptibility to proteolytic cleavage. Typical examples are fusions of a reporter polypeptide, e.g., luciferase, with a segment or domain of a ligand, e.g., a receptor-binding segment, so that the presence or location of the fused ligand may be easily determined. See, e.g., Dull, et al., U.S. Pat. No. 4,859,609. Other gene fusion partners include bacterial xcex2-galactosidase, trpE, Protein A, xcex2-lactamase, alpha amylase, alcohol dehydrogenase, and yeast alpha mating factor. See, e.g., Godowski, et al. (1988) Science 241:812-816.
Fusion peptides will typically be made by either recombinant nucleic acid methods or by synthetic polypeptide methods. Techniques for nucleic acid manipulation and expression are described generally, e.g., in Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.), vols. 1-3, Cold Spring Harbor Laboratory; and Ausubel, et al. (eds. 1993) Current Protocols in Molecular Biology, Greene and Wiley, NY. Techniques for synthesis of polypeptides are described, e.g., in Merrifield (1963) J. Amer. Chem. Soc. 85:2149-2156; Merrifield (1986) Science 232: 341-347; and Atherton, et al. (1989) Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford; and Grant (1992) Synthetic Peptides: A User""s Guide, W. H. Freeman, NY.
This invention also contemplates the use of derivatives of these Jak3 or xcex3 muteins other than mere variations in amino acid sequence or glycosylation. Such derivatives may involve covalent or aggregative association with chemical moieties. Covalent or aggregative derivatives will be useful as immunogens, as reagents in immunoassays, or in purification methods such as for affinity purification of receptors or other binding ligands. A Jak3 or xcex3c mutein can be immobilized by covalent bonding to a solid support such as cyanogen bromide-activated SEPHAROSE, by methods which are well known in the art, or adsorbed onto polyolefin surfaces, with or without glutaraldehyde cross-linking, for use in the assay or purification of anti-kinase antibodies or its receptor. The Jak3 or xcex3c muteins can also be labeled with a detectable group, for use in diagnostic assays. Purification of Jak3 or xcex3c muteins may be effected by immobilized antibodies or receptor.
The present invention contemplates corresponding muteins the isolation of additional closely related species variants, e.g., rodents, lagomorphs, carnivores, artiodactyla, perissodactyla, and primates.
The invention also provides means to isolate a group of related muteins displaying both distinctness and similarities in structure, expression, and function. Elucidation of many of the physiological effects of the muteins will be greatly accelerated by the isolation and characterization of distinct species variants.
The isolated genes encoding muteins will allow transformation of cells lacking expression of a corresponding Jak3 or xcex3c protein, e.g., either species types or cells which exhibit negative background activity.
Dissection of critical structural elements which effect the various receptor mediated functions provided by kinase binding is possible using standard techniques of modern molecular biology, particularly in comparing members of the related class. See, e.g., the homolog-scanning mutagenesis technique described in Cunningham, et al. (1989) Science 243:1339-1336; and approaches used in O""Dowd, et al. (1988) J. Biol. Chem. 263:15985-15992; and Lechleiter, et al. (1990) EMBO J. 9:4381-4390.
IV. Nucleic Acids
The described peptide sequences are readily made by expressing a DNA clone encoding the mutein, e.g., modified from a natural source, or a synthetic gene. The synthetic gene may be based upon a preferred codon usage, e.g., for production in bacteria. A number of different approaches should be available to successfully produce a suitable nucleic acid clone.
The purified protein or defined peptides are useful as described above. Synthetic peptides or purified protein can be presented to an immune system to generate monoclonal or polyclonal antibodies which recognize specifically the muteins. See, e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene; and Harlow and Lane (1989) Antibodies: A Laboratory Manual, Cold Spring Harbor Press.
This invention contemplates use of isolated DNA or fragments to encode a biologically active corresponding mutein. In addition, this invention covers isolated or recombinant DNA which encodes a biologically active antagonist or partial agonist protein or polypeptide.
An xe2x80x9cisolatedxe2x80x9d nucleic acid is a nucleic acid, e.g., an RNA, DNA, or a mixed polymer, which is substantially separated from other components which naturally accompany a native sequence, e.g., ribosomes, polymerases, and flanking genomic sequences from the originating species. The term embraces a nucleic acid sequence which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biologically synthesized by heterologous systems. A substantially pure molecule includes isolated forms of the molecule. Generally, the nucleic acid will be in a vector or fragment less than about 50 kb, usually less than about 30 kb, typically less than about 10 kb, and preferably less than about 6 kb.
An isolated nucleic acid will generally be a homogeneous composition of molecules, but will, in some embodiments, contain minor heterogeneity. This heterogeneity is typically found at the polymer ends or portions not critical to a desired biological function or activity.
A xe2x80x9crecombinantxe2x80x9d nucleic acid is defined either by its method of production or its structure. In reference to its method of production, e.g., a product made by a process, the process is use of recombinant nucleic acid techniques, e.g., involving human intervention in the nucleotide sequence, typically selection or production. Alternatively, it can be a nucleic acid made by generating a sequence comprising fusion of two fragments which are not naturally contiguous to each other, but is meant to exclude products of nature, e.g., naturally occurring mutants. Thus, for example, products made by transforming cells with any unnaturally occurring vector is encompassed, as are nucleic acids comprising sequence derived using any synthetic oligonucleotide process. Such is often done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site.
Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a single genetic entity comprising a desired combination of functions not found in the commonly available natural forms. Restriction enzyme recognition sites are often the target of such artificial manipulations, but other site specific targets, e.g., promoters, DNA replication sites, regulation sequences, control sequences, marker or purification tags, or other useful features may be incorporated by design. A similar concept is intended for a recombinant, e.g., fusion, polypeptide. Specifically included are synthetic nucleic acids which, by genetic code redundancy, encode polypeptides similar to fragments of these antigens, and fusions of sequences from various different species variants.
A significant xe2x80x9cfragmentxe2x80x9d in a nucleic acid context is a contiguous segment of at least about 17 nucleotides, generally at least about 22 nucleotides, ordinarily at least about 29 nucleotides, more often at least about 35 nucleotides, typically at least about 41 nucleotides, usually at least about 47 nucleotides, preferably at least about 55 nucleotides, and in particularly preferred embodiments will be at least about 60 or more nucleotides. Of particular interest are a plurality of distinct, e.g., nonoverlapping, segments of specified length. Typically, the plurality will be at least two, more usually at least three, and preferably 5, 7, or even more. While the length minima are provided, longer lengths, of various sizes, may be appropriate, e.g., one of length 7, and two of length 12.
Recombinant clones derived from genomic sequences, e.g., containing introns, will be useful for transgenic studies, including, e.g., transgenic cells and organisms, and for gene therapy. See, e.g., Goodnow (1992) xe2x80x9cTransgenic Animalsxe2x80x9d in Roitt (ed.) Encyclopedia of Immunology, Academic Press, San Diego, pp. 1502-1504; Travis (1992) Science 256:1392-1394; Kuhn, et al. (1991) Science 254:707-710; Capecchi (1989) Science 244:1288; Robertson (ed. 1987) Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, IRL Press, Oxford; and Rosenberg (1992) J. Clinical Oncology 10:180-199.
Substantial homology in the nucleic acid sequence comparison context means either that the segments, or their complementary strands, when compared, are identical when optimally aligned, with appropriate nucleotide insertions or deletions, in at least about 50% of the nucleotides, generally at least about 58%, ordinarily at least about 65%, often at least about 71%, typically at least about 77%, usually at least about 85%, preferably at least about 95 to 98% or more, and in particular embodiments, as high as about 99% or more of the nucleotides. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions, to a strand, or its complement, typically using a sequence encoding a mutein. Hybridization under stringent conditions should give a background of at least 2-fold over background, preferably at least 3-5 or more.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
Optical alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needlman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat""l Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., supra).
One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle (1987) J. Mol. Evol. 35:351-360. The method used is similar to the method described by Higgins and Sharp (1989) CABIOS 5:151-153. The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. For example, a reference sequence can be compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
Another example of algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described Altschul, et al. (1990) J. Mol. Biol. 215:403-410. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http:www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul, et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Nat""l Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.
In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Nat""l Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
A further indication that two nucleic acid sequences of polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions, as described below.
V. Antibodies
Antibodies can be raised to portions of Jak3 or xcex3c polypeptides and which bind specifically or selectively to the muteins described herein, including species or allelic variants, and fragments thereof. Additionally, antibodies can be raised to Jak3 or xcex3c muteins in either their active forms or in their inactive forms. Anti-idiotypic antibodies are also contemplated.
Antibodies, including binding fragments and single chain versions, against predetermined fragments of the ligands can be raised by immunization of animals with conjugates of fragments with immunogenic proteins. Monoclonal antibodies are prepared from cells secreting the desired antibody. These antibodies can be screened for binding to fragments containing sequences including the specified modifications. These monoclonal antibodies will usually bind with at least a KD of about 1 mM, more usually at least about 300 xcexcM, typically at least about 100 xcexcM, more typically at least about 30 xcexcM, preferably at least about 10 xcexcM, and more preferably at least about 3 xcexcM or better.
The antibodies of this invention can also be useful in diagnostic applications. See e.g., Chan (ed. 1987) Immunolocy: A Practical Guide, Academic Press, Orlando, Fla.; Price and Newman (eds. 1991) Principles and Practice of Immunoassay, Stockton Press, N.Y.; and Ngo (ed. 1988) Nonisotopic Immunoassay, Plenum Press, N.Y.
Mutein fragments may be joined to other materials, particularly polypeptides, as fused or covalently joined polypeptides to be used as immunogens. A mutein or its fragments may be fused or covalently linked to a variety of immunogens, such as keyhole limpet hemocyanin, bovine serum albumin, tetanus toxoid, etc. See Microbiology, Hoeber Medical Division, Harper and Row, 1969; Landsteiner (1962) Specificity of Serological Reactions, Dover Publications, New York; Williams, et al. (1967) Methods in Immunology and Immunochemistry, vol. 1, Academic Press, New York; and Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH Press, NY, for descriptions of methods of preparing polyclonal antisera. Alternatively, muteins may be attached to other solid substrates, e.g., to immobilize or as a synthetic substrate for synthesis.
In some instances, it is desirable to prepare monoclonal antibodies from various mammalian hosts, such as mice, rodents, primates, humans, etc. Description of techniques for preparing such monoclonal antibodies may be found in, e.g., Stites, et al. (eds.) Basic and Clinical Immunology (4th ed.), Lange Medical Publications, Los Altos, Calif., and references cited therein; Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH Press; Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed.), Academic Press, New York; and particularly in Kohler and Milstein (1975) in Nature 256:495-497, which discusses one method of generating monoclonal antibodies. Other suitable techniques involve in vitro exposure of lymphocytes to the antigenic polypeptides or alternatively to selection of libraries of antibodies in phage or similar vectors. See, Huse, et al. (1989) xe2x80x9cGeneration of a Large Combinatorial Library of the Immunoglobulin Repertoire in Phage Lambda,xe2x80x9d Science 246:1275-1281; and Ward, et al. (1989) Nature 341:544-546. The polypeptides and antibodies of the present invention may be used with or without modification, including chimeric or humanized antibodies. Frequently, the polypeptides and antibodies will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents, teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulins may be produced, see Cabilly, U.S. Pat. No. 4,816,567; Moore, et al., U.S. Pat. No. 4,642,334; and Queen, et al. (1989) Proc. Nat""l Acad. Sci. USA 86:10029-10033.
The antibodies of this invention can also be used for affinity chromatography in isolating the Jak3 or xcex3c proteins or polypeptides. Columns can be prepared where the antibodies are linked to a solid support. See, e.g., Wilchek, et al. (1984) Meth. Enzymol. 104:3-55.
Antibodies raised against each mutein will also be useful to raise anti-idiotypic antibodies. These will be useful in detecting or diagnosing various immunological conditions related to expression of the respective antigens.
VI. Making Agonists and Antagonists
DNA which encodes the Jak3 or xcex3c proteins or fragments thereof can be obtained by chemical synthesis, screening cDNA libraries, or screening genomic libraries prepared from a wide variety of cell lines or tissue samples. See, e.g., Okayama and Berg (1982) Mol. Cell. Biol. 2:161-170; Gubler and Hoffman (1983) Gene 25:263-269; and Glover (ed. 1984) DNA Cloning: A Practical Approach, IRL Press, Oxford. Suitable sequences can be obtained from GenBank. Biological mutagenesis methods of libraries may be applied to prepare variant polypeptides.
This DNA can be mutated for expression in a wide variety of host cells for the synthesis of mutein or fragments which can in turn, e.g., be used to generate polyclonal or monoclonal antibodies; for binding or screening studies; for construction and expression of modified molecules; and for structure/function studies. Combinatorial methods may be applied to prepare variants.
Vectors, as used herein, comprise plasmids, viruses, bacteriophage, integratable DNA fragments, and other vehicles which enable the integration of DNA fragments into the genome of the host. See, e.g., Pouwels, et al. (1985 and Supplements) Cloning Vectors: A Laboratory Manual, Elsevier, N.Y.; and Rodriguez, et al. (eds. 1988) Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Buttersworth, Boston, Mass.
For purposes of this invention, DNA sequences are operably linked when they are functionally linked to each other. For example, DNA for a presequence or secretory leader is operably linked to a polypeptide if it is expressed as a preprotein or participates in directing the polypeptide to the cell membrane or in secretion of the polypeptide. A promoter is operably linked to a coding sequence if it controls the transcription of the polypeptide; a ribosome binding site is operably linked to a coding sequence if it is positioned to permit translation. Usually, operably linked means contiguous and in reading frame, however, certain genetic elements such as repressor genes are not contiguously linked but still bind to operator sequences that in turn control expression. See e.g., Rodriguez, et al., Chapter 10, pp. 205-236; Balbas and Bolivar (1990) Methods in Enzymology 185:14-37; and Ausubel, et al. (1993) Current Protocols in Molecular Biology, Greene and Wiley, NY.
Representative examples of suitable expression vectors include pCDNA1; pCD, see Okayama, et al. (1985) Mol. Cell Biol. 5:1136-1142; pMC1neo Poly-A, see Thomas, et al. (1987) Cell 51:503-512; and a baculovirus vector such as pAC 373 or pAC 610, see, e.g., Miller (1988) Ann. Rev. Microbiol. 42:177-199.
It will often be desired to express a mutein or polypeptide in a system which provides a specific or defined glycosylation pattern. See, e.g., Luckow and Summers (1988) Bio/Technology 6:47-55; and Kaufman (1990) Meth. Enzymol. 185:487-511.
The appropriate mutein, or a fragment thereof, may be engineered to be phosphatidyl inositol (PI) linked to a cell membrane, but can be removed from membranes by treatment with a phosphatidyl inositol cleaving enzyme, e.g., phosphatidyl inositol phospholipase-C. This releases the antigen in a biologically active form, and allows purification by standard procedures of protein chemistry. See, e.g., Low (1989) Biochim. Biophys. Acta 988:427-454; Tse, et al. (1985) Science 230:1003-1008; and Brunner, et al. (1991) J. Cell Biol. 114:1275-1283.
Once a particular mutein has been characterized, fragments or derivatives thereof can be prepared by conventional processes for synthesizing peptides. These include processes such as are described in Stewart and Young (1984) Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford, Ill.; Bodanszky and Bodanszky (1984) The Practice of Peptide Synthesis, Springer-Verlag, New York; and Bodanszky (1984) The Principles of Peptide Synthesis, Springer-Verlag, New York; Villafranca (ed. 1991) Techniques in Protein Chemistry II, Academic Press, San Diego, Calif.; and Coligan, et al. (eds. 1996 and periodic supplements) Current Protocols in Protein Science John Wiley and Sons, Inc., New York, N.Y. For example, an azide process, an acid chloride process, an acid anhydride process, a mixed anhydride process, an active ester process (for example, p-nitrophenyl ester, N-hydroxysuccinimide ester, or cyanomethyl ester), a carbodiimidazole process, an oxidative-reductive process, or a dicyclohexylcarbodiimide (DCCD)/additive process can be used. Solid phase and solution phase syntheses are both applicable to the foregoing processes. See also chemical ligation, e.g., Dawson, et al. (1994) Science 266:776-779, a method of linking long synthetic peptides by a peptide bond.
VII. Uses
The present invention provides reagents which will find use in therapeutic or diagnostic applications as described elsewhere herein, e.g., in the general description for developmental abnormalities, or below in the description of kits for diagnosis.
The Jak3 or xcex3c polypeptides, muteins, fragments thereof, and antibodies thereto, should be useful in the evaluation or quality control of Jak3 or xcex3c constructs or fragments. They may also be useful in vitro or in vivo screening or treatment of conditions associated with abnormal physiology or development, including abnormal proliferation, e.g., cancerous conditions, or degenerative conditions and severe infections due to reduced levels of T and natural killer (NK) cells, as well as hypogammaglobulinemia. The structural relationship of the Jak3 or xcex3c protein to other Jak kinases suggests the possibility of biological activities beyond the Severe Combined Immunodeficiencies described patients with specific mutations in either the xcex3c subunit of a cytokine receptor (e.g., IL-2, IL-4, IL-7, IL-9, and IL-15) or in the Janus kinase. In particular, modulation of Jak kinase activities should be useful in situations where the Jak kinase functions have been implicated, e.g., lymphoid cell development, immunological responses, inflammation, graft rejection, rheumatoid arthritis, autoimmunity, abnormal proliferation, regeneration, degeneration, and atrophy of responsive cell types. For example, a disease or disorder associated with abnormal expression or abnormal signaling by Jak3 or xcex3c protein (e.g., SCID, XSCID, inhibition of T cell proliferation, or similar phenotypes, including those described in Candotti, et al. (1998) Springer Semin. Immunopathol. 19:401-415; Macchi, et al. (1995) Nature 377:65-68; Russell (1994) Science: 270:797-799; Morelon, et al. (1996) Blood 88:1708-1717; or Schmalstieg, et al. (1995) J. Clin. Invest. 95:1169-1173; also in developmental defects of lymphoid development as described in Park, et al. (1995) Immunity 3:771-782; Thomis, et al. (1995) Science 270:794-797; Macchi, et al., (1995) Nature 377:65-68; and Nosaka, et al. (1995) Science 270:800-802) besides the recognized effects, should be a potential target for treatment using an antagonist or agonist. The similarity in structures and mechanisms suggest potential hematopoietic or immunological functions may also exist.
Recombinant or synthetic Jak3 or xcex3c polypeptide muteins or, in some instances, antibodies can be purified and then administered to a patient. These reagents can be combined for therapeutic use with additional active or inert ingredients, e.g., in conventional pharmaceutically acceptable carriers or diluents, e.g., xcex3c variants, STAT5b, along with physiologically innocuous stabilizers and excipients. They may be combined with other antagonists, e.g., other cytokine antagonists, antibodies, mutein ligands, etc. These combinations can be sterile filtered and placed into dosage forms as by lyophilization in dosage vials or storage in stabilized aqueous preparations. This invention also contemplates use of antibodies or binding fragments thereof, including forms which are not complement binding.
The quantities of reagents necessary for effective therapy will depend upon many different factors, including means of administration, target site, physiological state of the patient, and other medicants administered. Thus, treatment dosages should be titrated to optimize safety and efficacy. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of these reagents. Animal testing of effective doses for treatment of particular disorders will provide further predictive indication of human dosage. Various considerations are described for the actual dosage of reagent, formulation or composition that modulates a disorder as described herein since the dosage depends on many factors, including the size and health of an organism, however one of one of ordinary skill in the art can use the following teachings describing the methods and techniques for determining clinical dosages. See Spilker (1984) Guide to Clinical Studies and Develoting Protocols Raven Press Books, Ltd., New York, pp. 7-13, 54-60; Spilker (1991) Guide to Clinical Trials Raven Press, Ltd., New York, pp. 93-101; Craig and Stitzel (eds. 1986) Modern Pharmacology, 2d ed., Little, Brown and Co., Boston, pp. 127-133; Speight (ed. 1987) Avery""s Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics, 3d ed., Williams and Wilkins, Baltimore, pp. 50-56; Tallarida, et al. (1988) Principles in General Pharmacology, Springer-Verlag, New York, pp. 18-20; Gilman, et al. (eds. 1990) Goodman and Gilman""s: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press; and Remington""s Pharmaceutical Sciences, cur. ed., Mack Publishing Co., Easton, Penn. Methods for administration are discussed therein and below, e.g., for oral, intravenous, intraperitoneal, or intramuscular administration, transdermal diffusion, and others. Pharmaceutically acceptable carriers will include water, saline, buffers, and other compounds described, e.g., in the Merck Index, Merck and Co., Rahway, N.J. Dosage ranges would ordinarily be expected to be in amounts lower than 1 mM concentrations, typically less than about 10 xcexcM concentrations, usually less than about 100 nM, preferably less than about 10 pM (picomolar), and most preferably less than about 1 fM (femtomolar), with an appropriate carrier. Slow release formulations, or a slow release apparatus will often be utilized for continuous administration. See, e.g., Langer (1990) Science 249:1527-533.
The Jak3 or xcex3c muteins may be administered directly to the host to be treated or, depending on the size of the compounds, it may be desirable to conjugate them to carrier proteins such as ovalbumin or serum albumin prior to their administration. Therapeutic formulations may be administered in many conventional dosage formulations. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical formulation. Formulations typically comprise at least one active ingredient, as defined above, together with one or more acceptable carriers thereof. Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient. Formulations include those suitable for oral, rectal, nasal, or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. See, e.g., Gilman, et al. (eds.) Goodman and Gilman""s: The Pharmacological Bases of Therapeutics, latest Ed., Pergamon Press; and Remington""s Pharmaceutical Sciences, latest ed., Mack Publishing Co., Easton, Pa.; Avis, et al. (eds. 1993) Pharmaceutical Dosage Forms: Parenteral Medications, Dekker, N.Y.; Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms: Tablets, Dekker, N.Y.; and Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms: Disperse Systems, Dekker, N.Y. The therapy of this invention may be combined with or used in association with other agents.
The muteins of this invention are particularly useful in kits and assay methods which are capable of screening compounds for interactions with binding proteins. Interaction of Jak3 JH7/JH6 fragments, e.g., attached to a solid phase, with, e.g., xcex3c box1/box2 fragments, can be used as the basis for a screening assay. Synthetic or recombinant fragments can be made, and labeled. In, e.g., a 96 well microtiter format, the interaction of the partners can be determined in the presence of candidate blocking compounds or mutein fragments. The partners have sequences disclosed in SEQ ID NO: 1 and 2. Segments of the JH7/JH6 and box1/box2 regions have been defined. See, e.g., Frank, et al. (1995) J. Biol. Chem. 270,14776-14785; and Zhao, et al. (1995) J. Biol. Chem. 270:13814-13818; and Murakami, et al. (1991) Proc. Nat""l Acad. Sci. USA 88:11349-11353. The JH7/JH6 fragments are in the region roughly of 1-256, but defined functionally by those segments which are critical for interaction with the xcex3c box1/box2 fragments, which run roughly from about 263-315. Conversely, the box1/box2 fragments are roughly in the region of 263-315, and are the essential minimal segments for functional interaction with the JH7/JH6 segment of Jak3. However, further analysis, by the methods described herein, will more narrowly define the respective interacting segments.
Several methods of automating assays have been developed in recent years so as to permit screening of tens of thousands of compounds in a short period. See, e.g., Fodor, et al. (1991) Science 251:767-773, which describes means for testing of binding affinity by a plurality of defined polymers synthesized on a solid substrate.
For example, antagonists can normally be found once the partners have been structurally identified. Testing of potential antagonist compound libraries, including combinatorial chemistry libraries, is now possible, based upon an in vitro or in vivo assay, or upon binding protein interaction. In particular, new agonists and antagonists will be discovered by using screening techniques described herein.
One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant DNA molecules expressing the binding pair. Cells may be isolated which express a binding protein in isolation from any others. Such cells, either in viable or fixed form, can be used for standard binding assays. See also, Parce, et al. (1989) Science 246:243-247; and Owicki, et al. (1990) Proc. Nat""l Acad. Sci. USA 87:4007-4011, which describe sensitive methods to detect cellular responses.
Rational drug design may also be based upon structural studies of the molecular shapes of the agonists or antagonists and other effectors or analogs. Effectors may be other proteins which mediate other functions in response to ligand binding, or other proteins which normally interact with the receptor. One means for determining which sites interact with specific other proteins is a physical structure determination, e.g., x-ray crystallography or 2 dimensional NMR techniques. These will provide guidance as to which amino acid residues form molecular contact regions. For a detailed description of protein structural determination, see, e.g., Blundell and Johnson (1976) Protein Crystallography, Academic Press, New York. Drug screening using muteins or fragments thereof or chemical compound libraries can identify compounds having ability to interfere with partner interaction. Alternatively, compounds which block interaction of the Jak3 with xcex3c , e.g., Jak3 JH7/JH6 or xcex3c box1/box2 muteins, may be identified by screening. Two hybrid type interaction (see, e.g., Fields and Song (1989) Nature 340:245-246) can serve as the basis for screening a library of muteins for interfering capability. Subsequent biological assays can then be utilized to determine if the compound has intrinsic interfering activity and is therefore an antagonist in that it blocks the signal process.
VIII. Kits
This invention also contemplates use of the muteins of the invention, proteins, polypeptides, fragments thereof, and their fusion products in a variety of diagnostic kits and methods for diagnosing or screening for antagonists of the binding interactions of a Jak3 kinase with a xcex3c polypeptide. Typically the kit will have a means of sequestering either a defined mutein peptide (e.g., compartment, affixed to a substrate etc.) or a means of sequestering a partner or reagent which recognizes one, e.g., Jak3 or xcex3c receptor fragments or antibodies.
A kit for determining the binding affinity of a test compound to a binding protein or receptor would typically comprise a test compound; a labeled compound, for example a receptor or antibody having known binding affinity for the Jak kinase or its mutein; a source of mutein; and a means for separating bound from free labeled compound, such as a solid phase for immobilizing the mutein. Once compounds are screened, those having suitable binding affinity can be evaluated in suitable biological assays, as are well known in the art, to determine whether they act as antagonists.
Antibodies, including antigen binding fragments, specific for muteins or unique fragments are useful in diagnostic applications to detect the presence of the muteins. In certain circumstances, it will be useful to quantitate amounts of muteins in a sample. Diagnostic assays may be homogeneous (without a separation step between free reagent and antigen-ligand complex) or heterogeneous (with a separation step). Various commercial assays exist, such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), enzyme-multiplied immunoassay technique (EMIT), substrate-labeled fluorescent immunoassay (SLFIA), and the like. See, e.g., Van Vunakis, et al. (1980) Meth Enzymol. 70:1-525; Harlow and Lane (1980) Antibodies: A Laboratory Manual, CSH Press, NY.; and Coligan, et al. (eds. 1993) Current Protocols in Immunology, Greene and Wiley, NY. 
Anti-idiotypic antibodies may have similar use to diagnose presence of antibodies against a mutein, as such may be diagnostic of various abnormal states.
Frequently, the reagents for diagnostic assays are supplied in kits, so as to optimize the sensitivity of the assay. For the subject invention, depending upon the nature of the assay, the protocol, and the label, either labeled or unlabeled antibody or receptor, or labeled mutein is provided. This is usually in conjunction with other additives, such as buffers, stabilizers, materials necessary for signal production such as substrates for enzymes, and the like. Preferably, the kit will also contain instructions for proper use and disposal of the contents after use. Typically the kit has compartments for each useful reagent. Desirably, the reagents are provided as a dry lyophilized powder, where the reagents may be reconstituted in an aqueous medium providing appropriate concentrations of reagents for performing the assay.
Any of the aforementioned constituents of the drug screening and the diagnostic assays may be used without modification or may be modified in a variety of ways. For example, labeling may be achieved by covalently or non-covalently joining a moiety which directly or indirectly provides a detectable signal. In many of these assays, the test compound, mutein, or antibodies thereto can be labeled either directly or indirectly. Possibilities for direct labeling include label groups: radiolabels such as 125I, enzymes (U.S. Pat. No. 3,645,090) such as peroxidase and alkaline phosphatase, and fluorescent labels (U.S. Pat. No. 3,940,475) capable of monitoring the change in fluorescence intensity, wavelength shift, or fluorescence polarization. Possibilities for indirect labeling include biotinylation of one constituent followed by binding to avidin coupled to one of the above label groups.
There are also numerous methods of separating the bound from the free partner, or alternatively the bound from the free test compound. A mutein can be immobilized on various matrixes followed by washing. Suitable matrixes include plastic such as an ELISA plate, filters, and beads. See, e.g., Coligan, et al. (eds. 1993) Current Protocols in Immunology, Vol. 1, Chapter 2, Greene and Wiley, NY. Other suitable separation techniques include, without limitation, the fluorescein antibody magnetizable particle method described in Rattle, et al. (1984) Clin. Chem. 30:1457-1461, and the double antibody magnetic particle separation as described in U.S. Pat. No. 4,659,678.
Methods for linking proteins or their fragments to the various labels have been extensively reported in the literature and do not require detailed discussion here. Many of the techniques involve the use of activated carboxyl groups either through the use of carbodiimide or active esters to form peptide bonds, the formation of thioethers by reaction of a mercapto group with an activated halogen such as chloroacetyl, or an activated olefin such as maleimide, for linkage, or the like. Fusion proteins will also find use in these applications.
Diagnostic kits which also test for the qualitative or quantitative presence of other markers are also contemplated. Diagnosis or prognosis may depend on the combination of multiple indications used as markers. Thus, kits may test for combinations of markers. See, e.g., Viallet, et al. (1989) Progress in Growth Factor Res. 1:89-97.
All references cited herein are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The broad scope of this invention is best understood with reference to the following examples, which are not intended to limit the invention to specific embodiments.